Peptide conjugated anti-cancer prodrugs

ABSTRACT

The present invention relates to prodrug molecules comprising conjugates of an antiproliferative drug, a protease specific cleavable peptide, and, optionally, a targeting peptide, with the prodrugs being substantially inactive prior to degradation of the cleavable sequence by proteolytic enzymes abundant within or in close proximity to the target cancer cell. Also, pharmaceutical compositions of the conjugates and the use of these compositions for treatment of cancer are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/555,939 filed Nov. 2, 2006, which is a continuation of U.S.application Ser. No. 10/382,240 filed Mar. 5, 2003, now U.S. Pat. No.7,135,547, which in turn is a continuation of International applicationPCT/IL01/00839 filed Sep. 5, 2001, which in turn claims the benefit of60/229,733 filed Sep. 5, 2000. The entire content of each application isexpressly incorporated herein by reference thereto.

FIELD OF INVENTION

The present invention relates to prodrug molecules comprising conjugatesof an antiproliferative drug, a protease specific cleavable peptide,and, optionally, a targeting peptide, said prodrugs being substantiallyinactive prior to degradation of the cleavable sequence by proteolyticenzymes abundant within or in close proximity to the target cancer cell,to pharmaceutical compositions comprising the conjugates and to the useof these compositions for treatment of cancer.

BACKGROUND OF THE INVENTION Chemotherapeutic Anti-Proliferative Drugs

Anti-proliferative drugs, also known as anti-metabolites, act byinhibiting crucial metabolic processes, and are commonly used in thetreatment of diseases involving abnormal cell proliferation, such astumors. However, the utility of these drugs is severely hampered bytheir excessive toxicity and adverse side effects on healthy cells ofthe treated patient. Therefore, it would be advantageous to be able toreduce these adverse effects by the use of a prodrug having decreasedtoxicity.

The use of prodrugs to impart desired characteristics such as increasedbioavailability or increased site-specificity of known drugs is arecognized concept in the state of the art of pharmaceuticaldevelopment. The use of various blocking groups, which must be removedin order to release the active drug is also known in the background art.Commonly, one or more blocking groups may be attached via an availableamine, hydroxyl group or other functional reactive group on the drug toyield an amide or an ester. This type of prodrug may be cleaved bynon-specific esterases to release the active principle in asustained-release fashion over a prolonged period of time compared tothe native drug species.

Methotrexate (MTX), for example, is an effective anti-proliferative drugcommonly used in cancer therapy. It is an analogue of dihydrofolate thatinhibits the enzyme dihydrofolate reductase (DHFR), thus depletingintracellular tetrahydrofolate (FH₄), which is an essential co-factorrequired for the de novo synthesis of purine nucleotides.

MTX, Mephalan and Chlorambucil are valuable drugs in the treatment ofmany rapidly growing tumors, however, their use is limited by thefrequency and severity of side effects. Unwanted side effects includetoxicity to all rapidly dividing normal cells, such as stem cells in thebone marrow, epithelial cells of the intestinal tract, hair folliclecells, etc.

Another major problem in chemotherapy, which is particularly relevant inthe case of anti-metabolites, is inherent or acquired resistance oftumors to cytotoxic drugs. For example, development of resistance to MTXfrequently follows prolonged exposure to this drug. Resistance may bedue to new mutations induced by the clinical treatment, or to positiveselection, by the treatment regimen, of pre-existing resistant mutantcell. Known mechanisms for development of resistance involve impairedtransport of MTX into cells, e.g. by mutations in the Reduced FolateCarrier (RFC), over expression of the target enzyme DHFR, or mutationsin the enzyme responsible for polyglutamination of reduced folates(FPGS).

A more severe problem in the clinic is the phenomenon of multi-drugresistance (MDR), which is a resistance to a broad spectrum ofstructurally unrelated cytotoxic drugs. MDR is mediated by transmembrane“pumps”, which actively expel chemotherapeutic drugs from the tumorcells. MDR significantly limits the efficacy of many cancer chemotherapyregimens and is a major factor in the failure of cancer chemotherapy.

It would, therefore, be most advantageous to have drug derivatives thatare specifically targeted to or selectively active in the diseased cellsrather than in the healthy cells, thus reducing undesirable sideeffects. It would also be desirable to generate new anti-proliferativeagents that overcome drug-resistance, as well as agents that are activeas cytotoxic drugs but lack or have a reduced ability to provoke MDRphenotype.

For specific cytotoxic drugs it has been suggested that the therapeuticindex of such drugs might be increased if the drug is covalently boundto a peptide that would be cleaved in the vicinity of the tumor cells bythe action of certain proteases. This approach has been suggested forpeptide conjugated Methotrexate (Kuefner et al., 1989) and forArabinofuranosyl cytosine (ara-C) lipid-peptide-drug conjugates (Mengeret al., 1994).

Glycosaminoglycans Binding Proteins

Many different types of cell surface polypeptides or glycoproteins havebeen utilized for targeting drugs to malignant cells, with variousdegrees of success.

The use of specific cell surface complex sugars as cell surface markersis much less well developed. In part this is due to the fact that theexpression of these structures cannot be followed in terms of genetranscription. In other words, the complex sugars are the product ofvarying expression of the glycosylation enzymes, and cannot be traceddirectly as gene products.

Proteoglycans are composed of long, unbranched sugar polymers, calledglycosaminoglycans (GAGs), which are covalently linked to a coreprotein. The proteoglycans constitute the extracellular matrix, such asthe cartilage, the basement membranes, and the connective tissue. Theyare also found on the cell surface (Bernfield, M. et al. 1992).Virtually all epithelia express cell-surface proteoglycans, representedprincipally by glypicans and syndecans. Glypicans are glycosylphosphatidyl inositol (GPI)-linked molecules, and bearglycosaminoglycans exclusively of the heparan sulfate type. Syndecansare transmembrane proteins, and are decorated with chondroitin sulfateand with heparan sulfate polymers. Syndecans exhibit a complex patternof cell and development specific expression, however, the molecularmechanisms responsible for this expression have not been fully explored.It was shown, for example that during wound healing the expression ofsyndecan-1 and -4 is induced. In the case of glypicans, it was shownthat glypican-1 is strongly expressed in human pancreatic cancer,whereas its expression is low in normal pancreas.

A variety of regulatory proteins bind tightly to GAGs, including growthfactors, cytokines, chemokines, extracellular matrix proteins, celladhesion molecules, lipid binding proteins, enzymes, and bloodcoagulation factors. The role of heparan sulphate proteoglycans (HSPGs)in growth factor signaling has been best characterized with respect tofibroblast growth factors (FGFs), which require the presence of heparansulfate for high affinity binding to their tyrosine kinase receptors(Yayon, A., et al. 1991). Several other growth factors have been foundto exhibit a strong requirement for a HSPG co-receptor in theirsignaling. These include heparin binding EGF-like growth factor(HB-EGF), hepatocyte growth factor (HGF), vascular endothelial growthfactor (VEGF) (Yamada, Y. et al., 1997), PDGF, TGF-beta, and other typesof growth factors.

Vascular endothelial growth factors (VEGFs) are mitogens for endothelialcells and are potent angiogenic factors in vivo. VEGF-165 contains thepeptide encoded by exon-7 of the VEGF gene, confers on VEGF-165 theability to bind heparan-sulfate molecules. VEGF-145 contains the peptideencoded by exon-6a of the VEGF gene, enabling VEGF-145 to bind ECM(Poltorak et al., 1997).

Several VEGF tyrosine-kinase receptor types have been characterized,these receptors mediates the mitogenic activity and induced cellmigration of VEGF. Other VEGF receptors, neuropilin-1 and neuropilin-2(Gitay-Goren, H., et al., 1992) bind only to the GAG binding forms ofVEGF (VEGF-165, VEGF-145) that have GAG binding peptides (axons 6a or 7)of the VEGF gene. These receptors are highly expressed in cancer cellssuch as human melanoma and carcinoma, but not expressed in normalmelanocytes.

VEGFs play a critical role in the process of tumor angiogenesis. Thisprocess is essential for tumor progression and for the subsequentprocess of tumor metastasis.

VEGF soluble receptors have been suggested as an inhibitor ofendothelial cell induced proliferation and angiogenesis (Kendall et al.U.S. Pat. No. 5,712,380).

Among the chemokines that are known to bind to heparin the bettercharacterized are Platelet factor 4 (PF4) (Morgan et al., 1977). PF4 isan anti-angiogenic factor that belongs to the CXC Chemokines family. PF4binds to several receptors that belong to the CXC receptor (CXCR) familyinvolved in angiogenesis. Kaposi's sarcoma cancer is indicated byuncontrolled angiogenesis that is associated with KSHV (Kaposi's sarcomaassociated herpes virus) that produces the CXCR-2 receptor homolog.

Injection of fluorescent PF4 to hamsters showed concentration of PF4 atcapillary endothelial cells at sites of active angiogenesis. PF4 isaccumulated at high concentrations in extra cellular matrix and basementmembrane due to its GAG binding ability.

PF4 can bind cell surface proteoglycans, and can be accumulated in theintracellular compartments (Neufeld at al., personal communication).Peptide from its GAG binding domain inhibited melanoma tumor growth inmice xenograft, though it had no effect on cancer cells in-vitro. CXCchemokines have been suggested as therapeutic molecules in modulatingthe angiogenic and angiostatic responses (U.S. Pat. No. 5,871,723).

Proteolytic Enzymes and Cancer Cells

Cancer invasion involves a proteolytic degradation of extracellularmatrix in the surrounding normal tissue. Excess matrix degradation isone of the hallmarks of cancer, and is an important component of theprocess of tumor progression (Fidler, I. J., 1997). In order forinvasion and metastasis to occur, the tumor cell must bypass thebasement membrane by degrading the components of the ECM.

Various proteases, in particular the serine protease plasmin, and avariety of matrix metalloproteinases (MMPs), have been implicated intumor invasion. Plasmin is formed from the inactive zymogen plasminogenby the plasminogen-activators. Plasminogen is produced in the liver andis present extracellulalrly throughout the body. One of theplasminogen-activators, the urokinase plasminogen activator (uPA), issynthesized as a pro-uPA that binds with high affinity to acell-surface-bound receptor, the uPA receptor (uPAR). Receptor bindingof pro-uPA strongly enhances the overall reaction leading to plasminformation (Dano, K. et al., 1994). Clinical findings have shown thatthere are elevated tumor antigen levels of Plasminogen Activator (uPA,tPA) and its receptor uPAR in cancer cells and tumors and it plays arole in tumor invasion and metastasis (Koopman et al., 1998; Schmidt etal., 1997).

The MMPs comprise of a large family of over 20 proteins that can degradeall the known components of the extracellular matrix (Massova, I. et al.1998). MMPs were identified in the tissues surrounding invasive cancers,and show over expression in malignant tissues.

The human aspartic proteinases include cathepsin D, cathepsin E,pepsinogen A, pepsinogen C, and rennin (Taggart, R. T., 1992).Cathepsins D and E are significantly elevated in various cancers andmetastases, hence applied as tumor cell markers of epithelial cancers(Matsue, K. et al., 1996)

Nowhere in the background art is it taught or suggested that it ispossible to use peptides as drug carriers useful to target prodrugs totumors.

SUMMARY OF THE INVENTION

It is an object of this invention to target a drug to malignant cells.It is a further object of the present invention to provide prodrugs thatare selectively activated in or near malignant cells. It is still afurther object of the present invention to provide a technique fortreating a malignant tumor or metastatic cancer by selective activationof such prodrugs in or near malignant cells.

These and yet other objectives are accomplished by the compositionsaccording to the present invention, wherein at least one drug iscovalently coupled either directly or by means of an appropriate linkerto a peptide moiety, which is specifically cleavable by a protease,which is more abundant in malignant cells or secreted by malignant cellsmore than normal cells. These compositions are prodrugs, which arespecifically released near or in the malignant cells by the action ofsaid protease.

According to currently preferred embodiments these prodrugs furthercomprise a targeting moiety. More preferably the targetor is a peptidesequence covalently attached to the cleavable peptide.

Advantageously, these prodrugs may further comprise blocking groups orprotecting groups to prevent their digestion by nonspecific proteases.

Compositions according to the present invention are prodrugs that may berepresented schematically as follows:

Protecting Group-Targetor Peptide-Protease Specific CleavableSequence-Linker-Drug (Formula I)

wherein “protection group” denotes any appropriate blocking group on theN-, or -C terminal part, or on the side chain of the peptide sequence,which is capable of blocking the action of exopeptidases orendopeptidases; “targetor peptide” may be absent and denotes any peptidesequence capable of causing preferential accumulation of the prodrug ator near the malignant cells; “Protease specific cleavable sequence”denotes any peptide sequence which comprises a peptide bond cleavable bya specific protease, which is more abundant within or in proximity tothe malignant cells; “linker” may be absent and denotes any chemicalcompound present between the drug and the peptide which may be removedchemically, enzymatically or may decompose spontaneously; and “drug”denotes any cytotoxic, cytostatic or chemotherapeutic drug. It is to beunderstood that the prodrug according to the present invention isgenerally pharmacologically substantially inactive until the cytotoxicdrug is released from the prodrug.

In a further embodiment, compositions according to the present inventionmay consist of at least one drug conjugated to the protease cleavablesequence and to the targetor peptide. In another embodiment,compositions according to the present invention may consist of aplurality of drug molecules conjugated to at least one proteasecleavable sequence and targetor peptide. The plurality of drug moleculesmay be the same or different at each occurrence.

The present invention further provides pharmaceutical compositionscomprising as an active ingredient a prodrug according to the presentinvention. Such pharmaceutical compositions may be administered by anysuitable route of administration.

The technique for activation of the prodrug comprises the followingsteps:

a) specifically cleaving a peptide bond within the peptide moiety of theprodrug by a protease;b) digesting the remaining peptide moiety of the prodrug by means of anynonspecific exopeptidase activity; and in cases where the linker moietyis present,c) releasing the active drug from the linker moiety by decomposition ofthe drug-linker bond.

Currently more preferred embodiments of the invention comprise aprodrug, wherein the chemotherapeutic drug is selected from a groupconsisting of Melphalan, Methotraxate, Chlorambucil, Doxorubicin, and5-Fluorouracil.

Currently most preferred embodiments according to the invention comprisea prodrug selected from the group consisting of

Prodrug 1: GAG binding domain of PF4-Protease cleavage site-Melphalan;Prodrug 2: GAG binding domain of PF4-Protease cleavagesite-Methotraxate;Prodrug 3: GAG binding domain of PF4-Protease cleavagesite-Chlorambucil;Prodrug 6: GAG and extracellular binding domain of VEGF-Proteasecleavage site-Chlorambucil;Prodrug 7: GAG and extracellular binding domain of VEGF-Proteasecleavage site-Melphalan.

Currently more preferred embodiments of the invention comprise aprodrug, wherein the release peptide that is susceptible to proteolyticdegradation is selected from a group consisting of protease cleavagesites of MMP1, MMP9, cathepsin S, tPA, and uPA:

SEQ ID NO 2: Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro SEQ ID NO5: Ser-Pro-Gly-Arg-Val-Val-Arg-Gly SEQ ID NO 7: Val-Arg-Gly

Currently more preferred embodiments of the invention comprise aprodrug, wherein the targetor is selected from a group consisting of GAGbinding domain of PF4, and GAG and extracellular matrix binding domainof VEGF:

SEQ ID NO 1: Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser SEQ IDNO 4: Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Val.

Currently most preferred embodiments according to the invention comprisea prodrug selected from the group consisting of:

SEQ ID NO 3: Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser coupled tot-Butoxycarbonyl-N-Melphalan; SEQ ID NO 3:Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser coupled toDi-t-Butoxycarbonyl-N- Methotrexate; SEQ ID NO 3:Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser coupled toChlorambucil; SEQ ID NO 6:Ser-Pro-Gly-Arg-Val-Val-Arg-Gly-Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser- Trp-Ser-Val coupled toChlorambucil; SEQ ID NO 8:Val-Arg-Gly-Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Val coupled tot-Butoxycarbonyl-N-Melphalan.

While the present invention discloses prodrugs which comprise a targetorpeptide, the present invention further discloses prodrugs in which thetargetor peptide is absent. Thus, the present invention provides aprodrug comprising a conjugate of at least one antiproliferative drugmolecule coupled directly or via a linker to a peptide moiety, thepeptide moiety being specifically cleavable by an asparaginyl proteaseabundant in or secreted by malignant cells, thereby preferentiallyreleasing the antiproliferative drug molecule within or at the malignantcells by the action of the asparaginyl protease. It should be noted thatthe conjugate is pharmacologically inactive.

According to additional embodiments, the peptide moiety cleavable by theasparaginyl protease consists from two to about fourteen amino acidscomprising at least one asparagine. According to further embodiments,the peptide moiety cleavable by the asparaginyl protease consists of 3,alternatively 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids comprising atleast one asparagine. According to a certain embodiment, the asparaginylprotease is legumain. According to yet further embodiments, the peptidemoiety cleavable by legumain consists of up to 14 amino acids andcomprises the amino acid sequence X1-X2-X3, wherein X1 is selected fromthe group consisting of Pro, Thr, Asn, and Ala, X2 is selected from thegroup consisting of Thr, Pro, Asn, and Ala, and X3 is Asn, the peptideconsists up to 14 amino acids. According to still further embodiments,the peptide moiety cleavable by legumain consists of up to 14 aminoacids which comprise the amino acid sequence selected from the groupconsisting of Pro-Thr-Asn (SEQ ID NO:9); Pro-Asn-Asn (SEQ ID NO:10);Pro-Ala-Asn (SEQ ID NO:11); Pro-Pro-Asn (SEQ ID NO:12); Thr-Thr-Asn (SEQID NO:13); Thr-Asn-Asn (SEQ ID NO:14); Thr-Ala-Asn (SEQ ID NO:15);Thr-Pro-Asn (SEQ ID NO:16); Asn-Thr-Asn (SEQ ID NO:17); Asn-Asn-Asn (SEQID NO:18); Asn-Ala-Asn (SEQ ID NO:19); Asn-Pro-Asn (SEQ ID NO:20);Ala-Thr-Asn (SEQ ID NO:21); Ala-Asn-Asn (SEQ ID NO:22); Ala-Ala-Asn (SEQID NO:23); Ala-Pro-Asn (SEQ ID NO:24); Thr-Thr-Asn-Leu (SEQ ID NO:25);Thr-Thr-Asn-Ala (SEQ ID NO:26); Pro-Thr-Asn-Leu (SEQ ID NO:27);Pro-Thr-Asn-Ala (SEQ ID NO:28); Pro-Asn-Asn-Leu (SEQ ID NO:29);Pro-Asn-Asn-Ala (SEQ ID NO:30); Thr-Asn-Asn-Leu (SEQ ID NO:31); andThr-Asn-Asn-Ala (SEQ ID NO:32). According to yet further embodiments,the peptide moiety cleavable by legumain consists of two amino acids ofthe amino acid sequence selected from the group consisting of SEQ IDNO:33 to SEQ ID NO:38. According to a certain embodiment, the peptidemoiety cleavable by legumain consists of the amino acid sequence as setforth in SEQ ID NOs:9 to 11.

According to further embodiments, the antiproliferative drug is selectedfrom the group consisting of cyclophosphamide, chlorambucil, busulfan,Melphalan, Thiotepa, ifosphamide, Nitrogen mustard, methotrexate,5-Fluorouracil, cytosine arabinoside, 6-thioguanine, 6-mercaptopurine,doxorubicin, daunorubicin, idorubicin, nimitoxantron, dactinomycin,bleomycin, mitomycin, plicamycin, epipodophyllotoxins vincristin,vinblastin, vindestin, Etoposide, Teniposide, carmustin, lomustin,semustin, streptozocin, adrenocorticorticoids, estrogens, antiestrogens,progestins, aromatase inhibitors, androgens, antiandrogens, dacarbazin,hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplastin, andcarboplatin. According to certain embodiments, the antiproliferativedrug is selected from the group consisting of Melphalan, Methotrexate,Chlorambucil, Doxorubicin, and 5-fluorouracil.

According to yet further embodiments, the prodrug further comprises atleast one protecting group coupled to the peptide moiety, the protectinggroup capable of preventing digestion of said peptide moiety bynonspecific proteases. According to still further embodiments, theprotecting group is selected from the group consisting oft-butoxycarbonyl and 9-fluorenylmethoxycarbonyl.

The present invention further provides a method for producing a prodrug,the method comprising the step of linking at least one antiproliferativedrug molecule and a peptide moiety, the peptide moiety beingspecifically cleavable by an asparaginyl protease abundant in orsecreted by malignant cells. According to some embodiments, the peptidemoiety cleavable by the asparaginyl protease consists of from two toabout fourteen amino acids. According to a certain embodiment, theasparaginyl protease is legumain. According to yet further embodiments,the peptide moiety cleavable by legumain consists of two to 14 aminoacids comprising the amino acid sequence X1-X2-X3, wherein X1 isselected from the group consisting of Pro, Thr, Asn, and Ala, X2 isselected from the group consisting of Thr, Pro, Asn, and Ala, and X3 isAsn. According to still further embodiments, the peptide cleavable bylegumain consists of up to 14 amino acids comprising the amino acidsequence selected from the group consisting of SEQ ID NO:9 to SEQ IDNO:32. According to still further embodiments, the peptide moietycleavable by legumain consists of two amino acids of the amino acidsequence selected from the group consisting of SEQ ID NO:33 to SEQ IDNO:38. According to a certain embodiment, the peptide moiety cleavableby legumain consists of the amino acid sequence set forth in SEQ IDNOs:9 to 11.

According to some embodiments, the antiproliferative drug to be linkedto the peptide moiety cleavable by an asparaginyl protease is selectedfrom the group consisting of cyclophosphamide, chlorambucil, busulfan,Melphalan, Thiotepa, ifosphamide, Nitrogen mustard, methotrexate,5-Fluorouracil, cytosine arabinoside, 6-thioguanine, 6-mercaptopurine,doxorubicin, daunorubicin, idorubicin, nimitoxantron, dactinomycin,bleomycin, mitomycin, plicamycin, epipodophyllotoxins vincristin,vinblastin, vindestin, Etoposide, Teniposide, carmustin, lomustin,semustin, streptozocin, adrenocorticorticoids, estrogens, antiestrogens,progestins, aromatase inhibitors, androgens, antiandrogens, dacarbazin,hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplastin, andcarboplatin. According to certain embodiments, the antiproliferativedrug to be linked to the peptide moiety cleavable by an asparaginylprotease is selected from the group consisting of Melphalan,Methotrexate, Chlorambucil, Doxorubicin, and 5-fluorouracil.

According to some embodiments, the prodrug to be produced by the methodof the present invention further comprises a linker between theantiproliferative drug and the peptide. According to additionalembodiments, the prodrug further comprises a protecting group capable ofpreventing digestion of the peptide moiety by nonspecific proteases.According to particular embodiments, the protecting group is selectedfrom the group consisting of t-butoxycarbonyl and9-fluorenylmethoxycarbonyl.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a prodrug model that includes a targeting moiety (T), aprotease cleavable moiety (R), and a chemotherapeutic drug (D).

FIG. 2 shows a model of an extracellular prodrug activation thatincludes the targeting of a prodrug to a marker present on a cancercell, and the release of the chemotherapeutic drug from its carrier by aspecific extracellular degrading enzyme.

FIG. 3 depicts a model of an intracellular prodrug activation thatincludes the targeting of a prodrug to a marker present on a cancercell, the internalization of the prodrug-marker complex into the cell,and the release of the chemotherapeutic drug from its carrier by aspecific intracellular degrading enzyme.

FIG. 4A-B shows a comparison between cancer and normal cell death atincreasing concentrations of the prodrug 3.

FIG. 5A-B depicts a comparison between normal and cancer cells death atincreasing concentrations of the prodrug 1.

FIG. 6 shows an in-vitro release of Chlorambucil from its peptidecarrier (prodrug 3) by purified MMPs, or by the cancer cell conditionedmedium.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention prodrug conjugates are provided whichcomprise at least one anti-proliferative drug covalently bound to apeptide sequence comprising a peptide bond specifically cleavable by aprotease. The peptide sequence further comprises a targeting sequence,designed to increase the localization of the conjugate to the vicinityof the malignant cells. These prodrugs may further comprise linkermoieties between the drug and the peptide, and may still furthercomprise protecting groups or blocking groups attached to the peptide.

In the specification and in the claims the term “drug” denotes anypharmacologically active agent capable of arresting cell growth, orkilling the cell in which it is present and includes known cytotoxic,cytostatic or antiproliferative drugs such as are known in the art,exemplified by such compounds as:

-   -   Alkaloids: Docetaxel, Etoposide, Irinotecan, Paclitaxel,        Teniposide, Topotecan, Vinblastine, Vincristine, Vindesine.    -   Alkylating agents: Busulfan, Improsulfan, Piposulfan, Benzodepa,        Carboquone, Meturedepa, Uredepa, Altretamine,        triethylenemelamine, Triethylenephosphoramide,        Triethylenethiophosphoramide, Chlorambucil, Chloranaphazine,        Cyclophosphamide, Estramustine, Ifosfamide, Mechlorethamine,        Mechlorethamine Oxide Hcl, Melphalan, Novemebichin, Perfosfamide        Phenesterine, Prednimustine, Trofosfamide, Uracil Mustard,        Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine,        Semustine Ranimustine, Dacarbazine, Mannomustine, Mitobronitol,        Mitolactol, Pipobroman, Temozolomide.    -   Antibiotics and analogs: Aclacinomycins, Actinomycins,        Anthramycin, Azaserine, Bleomycins, Cactinomycin, Carubicin,        Carzinophilin, Cromomycins, Dactinomycins, Daunorubicin,        6-Diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Idarubicin,        Menogaril, Mitomycins, Mycophenolic Acid, Nogalamycine,        Olivomycins, Peplomycin, Pirarubicin, Plicamycin, Porfiromycin,        Puromycine, Streptonigrin, Streptozocin, Tubercidin, Zinostatin,        Zorubicin.    -   Antimetabolites: Denopterin, Edatrexate, Methotrexate,        Piritrexim, Pteropterin, Tomudex, Trimetrexate, Cladridine,        Fludarabine, 6-Mercaptopurine, Pentostatine Thiamiprine,        Thioguanine, Ancitabine, Azacitidine, 6-Azauridine, Carmofur,        Cytarabine, Doxifluridine, Emitefur, Floxuridine, Fluorouracil,        Gemcitabine, Tegafur;    -   Platinum complexes: Caroplatin, Cisplatin, Miboplatin,        Oxaliplatin;    -   Others: Aceglatone, Amsacrine, Bisantrene, Defosfamide,        Demecolcine, Diaziquone, Eflornithine, Elliptinium Acetate,        Etoglucid, Etopside, Fenretinide, Gallium Nitrate, Hdroxyurea,        Lonidamine, Miltefosine, Mitoguazone, Mitoxantrone, Mopidamol,        Nitracrine, Pentostatin, Phenamet, Podophillinic acid        2-Ethyl-Hydrazide, Procarbazine, Razoxane, Sobuzoxane,        Spirogermanium, Teniposide Tenuazonic Acid, Triaziquone,        2,2′,2″-Trichlorotriethylamine, Urethan.

In the specification and in the claims the term “protease specificsequence” denotes any peptide sequence comprising a sequence cleavableby a specific protease, and includes peptides of from about two to aboutfourteen amino acids comprising at least one site that is cleaved by aspecific protease. More preferred are peptide sequences comprising fromabout three to about twelve amino acids, as exemplified hereinbelow.

In the specification and in the claims the term “linker” denotes anychemical compound, which may be present between the drug moiety and thepeptide moiety of the prodrug. This linker may be removed from the drugby chemical means, by enzymatic means, or spontaneously. The linker maybe pharmacologically inert or may itself provide added beneficialpharmacological activity. The term “spacer” may also be usedinterchangeably as a synonym for linker.

In the specification and in the claims the term “protection group”denotes any appropriate blocking group on the N-, or -C terminal part,or on the side chain of the peptide sequence, which is capable ofblocking the action of exopeptidases or endopeptidases, such as are wellknown in the art.

The protection group may itself be pharmacologically inert or mayprovide added pharmacologically beneficial attributes to the conjugate.Most advantageously the protecting group will be lipophilic, therebyimproving the ability of the conjugate to penetrate into cells.

The prodrug includes several moieties: an optional targeting moiety,consisting of a targetor peptide that recognizes cancer cells ormalignant tissues, a protease specific cleavable moiety, consisting of acleavable sequence recognized by degrading enzymes that are moreabundant within or in proximity to the malignant cells, and achemotherapeutic drug (FIG. 1).

The prodrug may act on cancer cells and tissues by several concurrentmechanisms as follows: (i) The extracellular prodrug activation—theprodrug is targeted to a cancer cell, which displays a cancer specificmarker recognized by the targeting moiety (FIG. 2A), the targetingmoiety binds to the marker (FIG. 2B), the chemotherapeutic drug isreleased from the carrier by a specific extracellular degrading enzyme(FIG. 2C), and the chemotherapeutic drug attacks the cancer cell (FIG.2D); (ii) The intracellular prodrug activation—the prodrug is targetedto a cancer cell, which displays a cancer specific marker recognized bythe targeting moiety (FIG. 3A), the targeting moiety binds to the marker(FIG. 3B), the prodrug-marker complex are internalized into the cell(FIG. 3C), the chemotherapeutic drug is released from the carrier by aspecific intracellular degrading enzyme, and the chemotherapeutic drugattacks the cancer cell (FIG. 3D).

In prodrugs according to the invention, the drug could be placed ateither the N-terminal or C-terminal side of the peptide. The skilledartisan will be able to optimize the appropriate linkage and position ofthe drug moiety within the prodrug. Various concerns should be takeninto consideration to guide the artisan in this decision, such asselection of the peptide sequence, selection of the linker, selection ofthe position of attachment to the drug species, and requirementsconcerning host intracellular enzymes for drug activation.

The principles that apply to the selection of peptide, linker,attachment site, etc., will be detailed herein for exemplary compounds.The principles may be generalized as follows:

a) Selection of the peptide sequence: any peptide sequence that iscleavable by a protease that is more abundant within or in proximity tocancer cells may be suitable.

b) Selection of the linker: any chemical moiety that can serve as alinker between the peptide and the drug. The linker may be cleaved bychemical reaction, enzymatic reaction, or spontaneously. The linker mayalso serve for optimizing the specificity of the peptide-proteaseinteraction.

c) Selection of the position of attachment to the drug species: the drugmay be attached to either one or to both sides of the peptides,according to the peptidase activities that exists in the targeted cells.

d) Selection of the protecting group: The protecting group may be anychemical moiety that reduces the non-specific prodrug degradation (toactive or inactive compounds). Advantageously, the protecting group canbe a compound that increases the selectivity of the prodrug towards thecancer cells or tissues, or a compound that increases the permeabilityof the prodrug towards the cancer cells or tissues. According to asecond embodiment of the invention, the protecting group can itself bereplaced by a second drug or a second molecule of the same drug.

e) Selection of the drug: the drug can be any anti-proliferative,cytotoxic or cytostatic agent. It may be released in protected orunprotected form, i.e., it can itself be a prodrug. For instance, thetargetor moiety may be cleaved extracellularly and the resultantdrug-linker conjugate may still be a prodrug that releases the activedrug species intracellularly for effective treatment of oncogenesis.

Selection of the Chemotherapeutic Drug

Chemotherapeutic drugs have different ways in which they inhibit cancer.Chemotherapeutic drugs can damage the DNA template by alkylation, bycross-linking, or by double-strand cleavage of DNA. Other cancer drugscan block RNA synthesis by intercalation. Some agents are spindlepoisons, such as vinca alkaloids, or anti-metabolites that inhibitenzyme activity, or hormonal and anti-hormonal agents. Chemotherapeuticdrugs for targeting may be selected from various groups of agents,including but not limited to alkylating agents, antimetabolites,antitumor antibiotics, vinca alkaloids, epipodophyllotoxins,nitrosoureas, hormonal and antihormonal agents, and toxins.

Currently more preferred alkylating agents may be exemplified bycyclophosphamide, chlorambucil, busulfan, Melphalan, Thiotepa,ifosphamide, Nitrogen mustard.

Currently more preferred antimetabolites may be exemplified bymethotrexate, 5-Fluorouracil, cytosine arabinoside, 6-thioguanine,6-mercaptopurin.

Currently more preferred antitumor antibiotics may be exemplified bydoxorubicin, daunorubicin, idorubicin, nimitoxantron, dactinomycin,bleomycin, mitomycin, plicamycin.

Currently more preferred vinca alkaloids and epipodophyllotoxins may beexemplified by vincristin, vinblastin, vindestin, Etoposide, Teniposide.

Currently more preferred nitrosoureas may be exemplified by carmustin,lomustin, semustin, streptozocin.

Currently more preferred hormonal and antihormonal agents may beexemplified by adrenocorticorticoids, estrogens, antiestrogens,progestins, aromatas inhibitors, androgens, antiandrogens.

Additional preferred random synthetic agents may be exemplified bydacarbazin, hexamethylmelamine, hydroxyurea, mitotane, procarbazide,cisplastin, carboplatin.

Selection of the Targeting Sequence

The targeting sequences for the chemotherapeutic drug may be aglycosaminoglycan binding domain, or other binding domain found on thecancer cells or tissues.

In this invention the selection of a specific glycosaminoglycan bindingdomain as a targeting sequence was made by structure analysis ofspecific glycosaminoglycan chains present on the cancer cells ortissues. Since different cell types have been shown to synthesizeproteoglycans with different glycosaminoglycan structures and functions,such differences may be utilized for the selection of targetor peptide.

The selection of specific glycosaminoglycan binding domains fortargeting can be carried out by different means, for example, byscreening for native glycosaminoglycan binding domains capable ofinteracting with the specific glycosaminoglycans found on cancer cellsor tissues. Alternatively, this selection can be carried out byscreening for specific peptides (in peptide libraries) that interactwith the specific glycosaminoglycans found on cancerous cells ortissues.

Native specific glycosaminoglycan binding domains for targeting can beselected from glycosaminoglycan binding proteins, exemplified but notlimited to growth factors including but not limited to fibroblast growthfactors (1-23), epidermal growth factors, platelet derived growthfactors, vascular endothelial growth factors, cytokines and chemokinesincluding but not limited to interleukins, PF4, GRO-alpha, GRO-beta,GRO-gamma, extracellular matrix and cell adhesion proteins including butnot limited to fibronectin, collagen, laminin, thrombospondin,integrins, N-CAM, PECAM, CD44, lipid binding proteins including but notlimited to lipoprotein lipase, apolipoprotein B and E, LDL, enzymesincluding but not limited to acetylcholinesterase, GAG degradingenzymes, blood coagulation factors including but not limited toantithrombin III tissue factor, and other proteins including but notlimited to influenza virus, Diphteria toxin, prion proteins, some ofthem involve in various malignancies.

Selection of the Release Sequence

Cancer invasion involves a proteolytic degradation of the extracellularmatrix of the surrounding normal tissue. Excess matrix degradation isone of the hallmarks of cancer, and is an important component of theprocess of tumor progression (Fidler, I. J., 1997). When a tumor cellacquires the ability to invade and destroy a normal tissue, it is termedmalignant. The ability to form tumor metastases is characteristic ofhighly malignant cancers with poor clinical outcome. In order to invadeand metastasize, the tumor cell must bypass the basement membrane bydegrading the components of the extracellular matrix. During theinvasion, the tumor cell penetrates the basement membrane underlying thetumor. It then moves through more extracellular matrix to reach thecirculation either through the lymphatic- or through the blood vessels.The process of entering the blood stream is termed intravasation. Inorder to establish itself at a distant site, the cancer cell repeats theentire process in reverse. During this process of extravasation thetumor cell leaves the blood circulation, and penetrates the host tissue,again crossing through a basement membrane. If the tumor cells arecapable of growing in this unfamiliar environment, clinicallysignificant metastases are formed and can pose a threat to the life ofthe host.

Various proteases have been implicated in tumor invasion. In particular,the serine protease plasmin, the MMPs, and the aspartic proteinases havebeen shown to be involved in this process.

The MMPs comprise of a large family of over 20 proteins that can degradeall the known components of the extracellular matrix (Massova I. et al.1998). These proteinases demonstrate some selectivity such that anindividual MMP has the ability to degrade a particular subset of matrixproteins. MMPs were identified in the tissues surrounding invasivecancers, and show over expression in malignant tissues.

The human aspartic proteinases include cathepsin D, cathepsin E,pepsinogen A, pepsinogen C, and renin. Cathepsins D and E are significlyelevated in various cancers and metastases, hence applied as tumor cellmarkers of epithelial cancers.

In the present invention specific protease cleavable sequences wereselected by structure analysis of specific biodegradable sequences thatare degraded by proteases that are more abundant within or in proximityto the malignant cells. The proteases are grouped as follows:

Matrix metalloproteinases may be exemplified but not limited tocollagenases, gelatinases, stromelysins.

Aspartic proteases may be exemplified but not limited to cathepsin D,cathepsin E, pepsinogen A, pepsinogen C, renin.

Serine proteases may be exemplified but not limited to plasmin,tissue-type plasminogen activator (tPA), urokinase-type plasminogenactivator (uPA).

Cysteine proteases may be exemplified but not limited to cathepsin B,cathepsin L, cathepsin S.

Asparaginyl proteases may be exemplified but not limited to legumain.

The protease specific cleavable sequences can be selected by screeningfor native degradation substrates of proteases, which are more abundantwithin or in proximity to malignant cells. Alternatively, the proteasespecific cleavable sequences can be selected by screening for specificpeptides (in peptide libraries) that can be susceptible to a proteolyticdegradation by proteases, which are more abundant within or nearmalignant cells.

Native specific biodegradable sequences can be selected from the groupsof native substrates as listed below:

Matrix metalloproteinases substrates exemplified but not limited tocollagens, gelatins, fibronectin, elastin, laminin, proteoglycans,serpin, uPA.

Aspartic proteases substrates exemplified but not limited to Bioactivepeptides, Beta-amyloid precursor.

Serine proteases substrates exemplified but not limited to plasminogen,fibrin, PAR1 thrombin receptor, uPAR-1 (uPA receptor).

Cysteine proteases substrates exemplified but not limited to collagens.

Asparaginyl proteases substrates exemplified but not limited toantigenic proteins for MHC class II, proenzymes (see, for example, ChenJ. M. et al., J. Biol. Chem. 272: 8090-8098, 1997; and Dando P. M. etal., Biochem J. 339: 743-749, 1999).

Currently most preferred embodiments of the invention comprise aconjugate, wherein the chemotherapeutic drug is selected from a groupconsisting of Melphalan, Methotraxate, Chlorambucil, Doxorubicin,5-Fluorouracil, folic acid, and N—N′-Diethylaminobezoic acid.

Currently most preferred embodiments encompass the following conjugates:

Prodrug 1: GAG binding domain of PF4-Protease cleavage site-MelphalanProdrug 2: GAG binding domain of PF4-Protease cleavagesite-Methotraxate.Prodrug 3: GAG binding domain of PF4-Protease cleavagesite-Chlorambucil.Prodrug 4: GAG binding domain of PF4-Protease cleavage site-folic acid.Prodrug 5: GAG binding domain of PF4-Protease cleavagesite-N—N′-Diethylaminobezoic acid.Prodrug 6: GAG and extracellular binding damain of VEGF-Proteasecleavage site-Chlorambucil.Prodrug 7: GAG and extracellular binding damain of VEGF-Proteasecleavage site-MelphalanProdrug 8: Asparaginy Protease cleavage site-MethotraxateProdrug 9: Asparaginy Protease cleavage site-DoxorubicinProdrug 10: Asparaginy Protease cleavage site-5-Fluorouracil.

Currently most preferred embodiments of the invention comprise aprodrug, wherein the protease cleavable peptide that is susceptible toproteolytic degradation is selected from a group consisting of proteasecleavage sites of MMP1, MMP9, cathepsin S, tPA, uPA, and asparaginylproteases:

SEQ ID NO 2: Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro SEQ ID NO 5is: Ser-Pro-Gly-Arg-Val-Val-Arg-Gly SEQ ID NO 7 is: Val-Arg-Gly

Currently most preferred embodiments of the invention comprise aprodrug, wherein the targetor is selected from a group consisting of GAGbinding domain of PF4, and GAG and extracellular matrix binding domainof VEGF:

SEQ ID NO 1: Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser SEQ IDNO 4 is: Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Val

Currently the specific embodiments of the present invention consist ofprodrugs composed of:

SEQ ID NO 3: Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser coupled tot-Butoxycarbonyl-N-Melphalan; SEQ ID NO 3:Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser coupled toDi-t-Butoxycarbonyl-N- Methotrexate; SEQ ID NO 3:Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser coupled toChlorambucil; SEQ ID NO 6:Ser-Pro-Gly-Arg-Val-Val-Arg-Gly-Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser- Trp-Ser-Val coupled toChlorambucil; SEQ ID NO 8:Val-Arg-Gly-Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Val coupled tot-Butoxycarbonyl-N-Melphalan

According to some embodiments, the prodrug is devoid of a targetorpeptide, the asparaginyl protease of the prodrug is legumain, and thepeptide moiety cleavable by legumain consists of known amino acidsequences (see, for example, Chen J. M. et al., J. Biol. Chem. 272:8090-8098, 1997; and Dando P. M. et al., Biochem J. 339: 743-749, 1999).According to exemplary embodiments, the peptide cleavable by legumain isselected from the group consisting of:

Pro-Thr-Asn; (SEQ ID NO: 9) Pro-Asn-Asn; (SEQ ID NO: 10) Pro-Ala-Asn;(SEQ ID NO: 11) Pro-Pro-Asn; (SEQ ID NO: 12) Thr-Thr-Asn; (SEQ ID NO:13) Thr-Asn-Asn; (SEQ ID NO: 14) Thr-Ala-Asn; (SEQ ID NO: 15)Thr-Pro-Asn; (SEQ ID NO: 16) Asn-Thr-Asn; (SEQ ID NO: 17) Asn-Asn-Asn;(SEQ ID NO: 18) Asn-Ala-Asn; (SEQ ID NO: 19) Asn-Pro-Asn; (SEQ ID NO:20) Ala-Thr-Asn; (SEQ ID NO: 21) Ala-Asn-Asn; (SEQ ID NO: 22)Ala-Ala-Asn; (SEQ ID NO: 23) Ala-Pro-Asn; (SEQ ID NO: 24)Thr-Thr-Asn-Leu; (SEQ ID NO: 25) Thr-Thr-Asn-Ala; (SEQ ID NO: 26)Pro-Thr-Asn-Leu; (SEQ ID NO: 27) Pro-Thr-Asn-Ala; (SEQ ID NO: 28)Pro-Asn-Asn-Leu; (SEQ ID NO: 29) Pro-Asn-Asn-Ala; (SEQ ID NO: 30)Thr-Asn-Asn-Leu; (SEQ ID NO: 31) and Thr-Asn-Asn-Ala. (SEQ ID NO: 32)

According to additional embodiments, the peptide cleavable by legumainconsists of two amino acids (see, for example, Chen J. M. et al., J.Biol. Chem. 272: 8090-8098, 1997; and Dando P. M. et al., Biochem J.339: 743-749, 1999). The amino acid sequence of the peptide cleavable bylegumain is thus selected from the group consisting of:

Asn-Lys; (SEQ ID NO: 33) Asn-Leu; (SEQ ID NO: 34) Asn-Ala; (SEQ ID NO:35) Asn-Glu; (SEQ ID NO: 36) Asn-Asp; (SEQ ID NO: 37) and Asn-Asn. (SEQID NO: 38)

According to certain embodiments, the prodrug is composed of:

SEQ ID NO 9: Pro-Thr-Asn coupled to MethotrexateSEQ ID NO 10: Pro-Asn-Asn coupled to DoxorubicinSEQ ID NO 11: Pro-Ala-Asn coupled to 5-Fluorouracil.

Although the invention will now be described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

Example 1 Synthesis of t-Butoxycarbonyl-N-Melphalan

100 mg Melphalan were dissolved in 0.56 ml water solution of NaOH (0.44g in 11 ml HPLC water). The solution was mixed and 0.25 ml of t-butylalcohol was added. 0.08 ml of di-tert-butylcarbonate was added to themixture and mixed for 1.5 hours.

The product t-Butoxycarbonyl-Melphalan was extracted by Hexane andprecipitated by 0.1N HCl. The precipitate was washed with HPLC water anddissolved in Ethanol. Purified t-Butoxycarbonyl-N-Melphalan wascrystallized from Ethanol.

Purity of t-Butoxycarbonyl-N-Melphalan was analyzed using reversed-phasehigh performance liquid chromatography (HPLC) on LiChroCART 250-4 HPLCcartridge Purospher RP-18 (purchased from Merck).

The molecular weight and chemical structure of the product were analyzedby Mass spectrometry on matrix assisted laser desorption ionization(MALDI) or electrospray ionization (ESI), interfaced to quadruple iontrap and TOF (time of flight) mass spectrometer.

Example 2 Synthesis of 9-Fluorenylmethoxycarbonyl-N-Melphalan

0.5 mmol Melphalan and 0.7 mmol 9-fluorenylmethel-succinimidyl-carbonatewere dissolved in 5 ml of acetonitrile/water solution (2/1). Thesolution was mixed and 0.1 ml of diisopropylethylamine was added. Themixture was stirred for 20 hours.

Acetonitrile was evaporated. The product9-fluorenylmethelcarbonyl-Melphalan was extracted by Ethylacetate/5%citric acid. The organic layer was washed with 5% citric acid and thenevaporated to dryness. The product was dissolved in Ethanol. Purified9-fluorenylmethelcarbonyl-N-Melphalan was crystallized from Ethanol.

Purity, molecular weight and chemical structure of9-fluorenylmethelcarbonyl-N-Melphalan were analyzed using reversed-phaseHPLC, and Mass spectrometry as described in Example 1.

Example 3 Synthesis of Di-t-Butoxycarbonyl-N-Methotrexate

0.5 mmol Methotrexate(N-[4-[[(2,4-diamino-6-pteridin-yl)-methylamino]benzoyl]-L-glutamicacid) and 1.5 mmol of triethylamine were dissolved in 5 ml HPLC water.The solution was mixed and 1.1 mmol ofS-Boc-2-mercapto-4,6-dimethyl-pirimidine dissolved in 5 ml of dioxanewas added. The reaction mixture was mixed for 18 hours. The productDi-t-Butoxycarbonyl-N-Methotrexate was extracted by Ethylacetate andsaturated with citric acid solution. The organic layer was washed withsaturated citric acid solution and saturated NaCl solution and thenevaporated to dryness. The product was dissolved in Ethanol. PurifiedDi-t-Butoxycarbonyl-N-Methotrexate was crystallized from Ethanol/icecold water solution.

Purity, molecular weight and chemical structure ofDi-t-Butoxycarbonyl-N-Methotrexate were analyzed using reversed-phaseHPLC, and Mass spectrometry as described in Example 1.

Example 4 Synthesis of Peptide Carrier I (T-R)

The peptide carrier for the chemotherapeutic drug was synthesized byusing combinatorial chemistry and solid phase peptide synthesis.Fmoc/Boc protected amino acids were used for the synthesis.

The first amino acid was bound to activated solid support such aspolystyrene beads onto which hydroxybenzyl alcohol linker has beenattached (Wang, 1973).

Synthesis of the peptide carrier I (SEQ ID NO 3) containing targeting(SEQ ID NO 1) and cleavable (SEQ ID NO 2) domains (T-R) was as follows:

Targeting (T) peptide sequence (SEQ ID NO 1) is:Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln- Ser Cleavable (R)peptide sequence (SEQ ID NO 2) is:Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro

The first amino acid Fmoc-Ser(But)-OH was coupled to Wang resin (Wang,1973)

By using Dichlorobenzoylchloride method: 1 g of Wang resin was swollenin 8 ml of dimethylformamid (DMF) for 30 minutes. The resin was washed 5times with DMF. Two equivalents of Fmoc-Ser(But)-OH were dissolved in 8ml DMF, and added to the resin. The resin was shaked for 30 minutes atroom temperature. 3.3 equivalents of Pyridine and 2 equivalents of2,6-Dichlorobenzoylchloride were added to the resin and the resin wasshaked for 20-24 hours. The resin was washed on a filter glass with DMFand 1,2-Dichloroethane (DCE). The remaining hydroxyl groups of the resinwere benzoylated with Benzoyl chloride (0.3 ml) and Pyridine (0.3 ml) in8 ml DCE for 2 hours at room temperature. The resin was washed on afilter glass with DMF and with methanol, and dried in vacuum over silicagel.

At the end of the coupling the Fmoc-Ser(But)-Resin was deprotected bypiperidine method: 1 g resin with 0.5 mmol Fmoc-amino acid wasdeprotected 4 times with 10 ml of 20%-50% Piperidine in NMP or DMF.Deprotection was monitored by spectrophotometer measuring the level ofthe free Fmoc residue at 290 nm of each deprotection step. At the end ofdeprotection the resin was washed 10 times with DMF and methanol, and asample of the resin beads was analyzed by Kaiser test (Kaiser et al.,1970).

The next amino acids were bound to the peptide-resin by subsequent stepsof deprotection and coupling made for the extension of the peptide onthe resin using DIC/HOBt method: Fmoc-amino acid was dissolved in NMP orDMF for 3 minutes and reactivated with DIC/HOBt (molar ratio 1:1:1 toamino acid) for 20 minutes. Coupling of amino acid to peptide-resin wasmade for 60 minutes at room temperature with mechanical mixing andnitrogen bubbling.

At the end of peptide synthesis cleavage of the peptide from the resinwas made using TFA and scavengers (10-20 ml for 1 g peptide-resin) for1-4 hours mixing at room temperature. The eluate was filtered from theresin, the resin was washed 2 times with TFA, and the filtrates werecombined. Most of the TFA was evaporated from the elution, and peptidewas precipitated from TFA using ice-cold diisopropyl-ether. The peptideprecipitate was washed 3 times with ice-cold diisopropyl ether, and thenevaporated. The peptide was dissolved in water or buffer andlyophilized.

The peptide was analyzed using reversed-phase HPLC, mass spectrometry,and gel electrophoresis separation.

HPLC was made on LiChroCART 250-4 HPLC cartridge LiChrospher WP 300RP-18 (5 micrometer) (purchased from Merck). The prodrug was separatedon HPLC using a gradient of 20% B to 100% B at flow rate of 0.5 ml/min,and the product was detected at 214 nm (Fluent A: water+0.1% TFA, FluentB: Acetonitrile+10% water+0.1% TFA).

Mass spectrometry was made by matrix assisted laser desorptionionization (MALDI) or electrospray ionization (ESI), interfaced toquadruple ion trap and TOF (time of flight) mass spectrometer.

Gel electrophoresis was made on Tris-Tricin/SDS 10%-20% gradient gels(purchased from Bio-Rad).

Example 5 Synthesis of Peptide Carrier II

Synthesis of the Peptide Carrier II (SEQ ID NO 6) Containing Targeting(SEQ ID NO 4) and Cleavable (SEQ ID NO 5) Domains (T-R)

Targeting (T) peptide sequence (SEQ ID NO 4) is:Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Val Cleavable (R) peptide sequence (SEQ ID NO 5)is: Ser-Pro-Gly-Arg-Val-Val-Arg-Gly

The first amino acid Fmoc-Val-OH was coupled to Wang resin (Wang, 1973)by using Dichlorobenzoylchloride method as described in example 4.

At the end of the coupling the Fmoc-Val-Resin was deprotected by DBUmethod: 1 g resin with 0.5 mmol Fmoc-amino acid was deprotected 4 timeswith 10 ml of 2% 1,8-diazabicyco[5.4.0]undec-7-ene (DBU) and 2%Piperidine in NMP or DMF. At the end of deprotection a sample of theresin beads was analyzed by Kaiser test.

The next amino acids were bound to the peptide-resin by subsequent stepsof deprotection and coupling made for the extension of the peptide onthe resin using DIC/HOBt method as described in Example 4 or HBTU/HOBtmethod: Fmoc-amino acid was dissolved in NMP or DMF for 3 minutes,reactivated with HOBt (molar ratio 1:1 to amino acid) for 3 minutes andtransferred to the peptide-resin. HBTU (molar ratio 1:1 to amino acid)was added for 5 minutes and DIPEA (molar ratio 2:1 to amino acid) wasadded. Coupling of amino acid to peptide-resin was made for 120 minutesat room temperature with mechanical mixing and nitrogen bubbling.

Peptide cleavage and side chain deprotection was made as described inexample 4.

The peptide was analyzed using reversed-phase HPLC, mass spectrometry,and gel electrophoresis separation as described in example 4.

Example 6 Synthesis of Peptide Carrier III

Synthesis of the Peptide Carrier III (SEQ ID NO 8) Containing Targeting(SEQ ID NO 4) and Cleavable (SEQ ID NO 7) Domains (T-R)

Targeting (T) peptide sequence (SEQ ID NO 4) is:Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Val Cleavable (R) peptide sequence (SEQ ID NO 7)is: Val-Arg-Gly

The first amino acid Fmoc-Val-OH was coupled to Rink resin. One gram ofRink resin was swollen in 10 ml of DMF for 1 hour. The resin was washed5 times with DMF. Resin was deprotected 4 times with 20% Piperidine inNMP. Deprotection was monitored by spectrophotometer measuring the levelof the free Fmoc residue at 290 nm. At the end of deprotection the resinwas washed 10 times with NMP, methanol, NMP, and a sample of the resinbeads was analyzed by Kaiser test (Kaiser et al 1970).

Amino acids were bound to the peptide-resin by subsequent steps ofdeprotection and coupling made for the extension of the peptide on theresin using HBTU/HOBt method or TBTU/HOBt method: Fmoc-amino acid wasdissolved in NMP or DMF for 3 minutes, reactivated with HOBt (molarratio 1:1 to amino acid) for 3 minutes and transferred to thepeptide-resin. TBTU (molar ratio 1:1 to amino acid) was added for 5minutes and DIPEA (molar ratio 3:1 to amino acid) was added. Coupling ofamino acid to peptide-resin was made for 120 minutes at room temperaturewith mechanical mixing and nitrogen bubbling.

At the end of peptide synthesis cleavage of the peptide from the resinwas made using 2 step cleavage method: 20%-50% TFA in DCM and scavengers(10-20 ml for 1 g peptide-resin) for 15-30 minutes mixing at roomtemperature. The elution was filtered from the resin, the resin waswashed 2 times with 20% TFA/DCM, and filtrates were combined. Most ofthe liquid was evaporated from the elution, and 95% TFA and 5%scavengers were added to the elute for 1-4 hours of deprotection.

Peptide was precipitated from TFA using ice-cold t-butyl-methyl-ether.The peptide precipitate was washed 3 times with ice-coldt-butyl-methyl-ether, and then evaporated. The peptide was dissolved inwater or buffer and lyophilized.

The peptide was analyzed using reversed-phase HPLC, mass spectrometry,and gel electrophoresis separation as described in Example 4.

Example 7 Synthesis of Prodrug 1 Peptide I-Melphalan

Peptide I (SEQ ID NO 3):Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln-Serwas synthesized as described in Example 4 and then coupled to the drug.

t-Butoxycarbonyl-N-Melphalan (molar ratio 1:2 to peptide) was dissolvedin DMF, and mixed for 5 minutes with the peptide-resin. TBTU (molar rate1:1) was added for 5 minutes and DIPEA (molar ratio 2:1) was added.Coupling of t-Butoxycarbonyl-N-Melphalan to peptide-resin was made for2-24 hours at room temperature with mechanical mixing and nitrogenbubbling. The coupling was monitored by spectroscopic measuring at 254nm of the free t-Butoxycarbonyl-N-Melphalan in the reaction mixture.

At the end of the prodrug 1 synthesis cleavage and side chaindeprotection of peptide-Melphalan was made as described in Example 4.

The peptide-Melphalan was analyzed using reversed-phase HPLC, massspectrometry, and gel electrophoresis separation as described in Example4.

Example 8 Synthesis of Prodrug 2-Peptide I-Methotrexate

Peptide I (SEQ ID NO 3):Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln-Serwas synthesized as described in Example 4 and then coupled to the drug.

Di-t-Butoxycarbonyl-N-Methotrexate (molar ratio 1:1.2 to peptide) wasdissolved in NMP, and mixed for 5 minutes with the peptide-resin. HBTU(molar ratio 1:1 to methotrexate) was added for 5 minutes and DIPEA(molar ratio 2:1 to methotrexate) was added. Coupling of methotrexate topeptide-resin was made for 2-24 hours at room temperature withmechanical mixing and nitrogen bubbling. The coupling was monitored byspectroscopic measuring at 302 nm of the free methotrexate in thereaction mixture.

At the end of the prodrug 2 synthesis, cleavage, and side chaindeprotection of peptide-Methotrexate were carried out as described inExample 4.

The peptide-Methotrexate was analyzed using reversed-phase HPLC, massspectrometry, and gel electrophoresis separation as described in Example4.

Example 9 Synthesis of Prodrug 3-Peptide I-Chlorambucil

Peptide I (SEQ ID NO 3):Tyr-Gly-Leu-Leu-Gly-Ile-Ala-Gly-Pro-Pro-Gly-Pro-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Gln-Serwas synthesized as described in Example 4 and then coupled to the drug.

Chlorambucil (molar ratio 1:2 to peptide) was dissolved in NMP, andmixed for 5 minutes with the peptide-resin. HBTU (molar ratio 1:1 tochlorambucil) was added for 5 minutes and DIPEA (molar rate 2:1 tochlorambucil) was added. Coupling of chlorambucil to peptide-resin wasmade for 2-24 hours at room temperature with mechanical mixing andnitrogen bubbling. The coupling was monitored by spectroscopic measuringat 263 nm of the free chlorambucil in the reaction mixture. At the endof the peptide-drug synthesis the resin was washed for 5 times with NMP,and 10 times with DCM on cinder glass. Cleavage of the drug from theresin was carried out by 90% TFA containing 5% H2O and 5%triisopropylsilan for 3 hours under mixing at room temperature. The drugwas worked-up using process 4.

The peptide-Chlorambucil was analyzed using reversed-phase HPLC, massspectrometry, and gel electrophoresis separation as described in Example4.

Example 10 Synthesis of Prodrug 4-Peptide I-Folic Acid

Peptide I (SEQ ID NO 3) was synthesized as described in Example 4 andthen coupled to folic acid. Folic acid was dissolved in DMF for 3minutes, reactivated with PyBOP (molar ratio 1:1.3 to acid) for 3minutes. DIPEA (molar rate 2:1 to acid) was added and the mixture wastransferred to the peptide-resin. Coupling of folic acid topeptide-resin was made for 120 minutes at room temperature withmechanical mixing and nitrogen bubbling.

Cleavage and analysis of peptide-folic acid was carried our as describedin example 8.

Example 11 Synthesis of Prodrug 5-Peptide I-N—N′-DiethylaminobenzoicAcid

Peptide I (SEQ ID NO 3) was synthesized as described in Example 4 andthen coupled to N—N′-Diethylaminobenzoic acid.

N—N′-Diethylaminobenzoic acid (molar ratio 1:2 to peptide) was dissolvedin NMP, and mixed for 5 minutes with the peptide-resin. HBTU (molarratio 1:1 was added for 5 minutes and DIPEA (molar rate 2:1 was added.Coupling of N—N′-Diethylaminobenzoic acid to peptide-resin was made for2-24 hours at room temperature with mechanical mixing and nitrogenbubbling. The coupling was monitored by spectroscopic measuring at 263nm of the free N—N′-Diethylaminobenzoic in the reaction mixture.

Cleavage and analysis of peptide-N—N′-Diethylaminobenzoic was carriedout as described in example 9.

Example 12 Synthesis of Prodrug 6-Peptide II-Chlorambucil

Synthesis of the peptide carrier II (SEQ ID NO 6):

Ser-Pro-Gly-Arg-Val-Val-Arg-Gly-Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Valwas synthesized as described in Example 5 and then coupled toChlorambucil as described in example 9.

Cleavage and analysis of peptide-chlorambucil was carried out asdescribed in example 9.

Example 13 Synthesis of Prodrug 7-Peptide III-Melphalan

Synthesis of the peptide carrier II (SEQ ID NO 8):

Val-Arg-Gly-Lys-Gly-Lys-Gly-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Trp-Ser-Valwas synthesized as described in Example 6 and then coupled to Melphalanas described in example 7.

Cleavage and analysis of peptide-Melphalan was carried out as describedin example 6.

Example 14 Synthesis of Legumain Cleavable Prodrug 8 A. Synthesis ofLegumain Cleavable Peptide: Pro-Thr-Asn

The peptide cleavable by legumain is synthesized by using combinatorialchemistry and solid or liquid phase peptide synthesis. Fmoc/Bocprotected amino acids are used for the synthesis.

The first amino acid Fmoc-Asn(Trt)-OH is coupled to Wang resin by usingDichlorobenzoylchloride method as described in example 4.

The next amino acids are bound to the peptide-resin by subsequent stepsof deprotection and coupling. Aat the end of peptide synthesis, cleavageof the peptide from the resin is performed by using TFA as described inexample 4.

The peptide is analyzed using reversed-phase HPLC, mass spectrometry,and spectrophotometer.

B. Synthesis of Boc-Pro-Thr-Asn-MTX

The peptide Pro-Thr-Asn is protected with Boc usingdi-tert-butylcarbonate method as described in example 1. Boc protectedpeptide is coupled to methotrexate (MTX) using DIC as follows:Boc-Pro-Thr-Asn is dissolved in DMF, DIC is added (molar ratio 1:1) andmixed for 30 minutes. Solid methotrexate is added to the mixture (molarratio 1:1 drug:peptide) and incubated for 24 hours at room temperaturewith mixing. The DMF is evaporated and the product is precipitated andwashed with tert-Butyl-Methyl-Ether.

C. Synthesis of Pro-Thr-Asn-MTX

Cleavage of the Boc group is carried out by 90% TFA containing 5% H2Ofor 3 hours under mixing at room temperature. The Thr-Thr-Asn-MTX isanalyzed using reversed-phase HPLC and mass spectrometry as described inExample 4. Pro-Thr-Asn-Methotrexate gives solid yellowish powder withmolecular mass of 766 g/mol.

Example 15 Synthesis of Legumain Cleavable Prodrug 9

A. Synthesis of Legumain cleavable peptide: Thr-Thr-Asn-Leu

The peptide cleavable by legumain is synthesized as described in example14.

B. Synthesis of Fmoc-Thr-Thr-Asn-Leu-Doxorubicin

The peptide is protected with Fmoc using9-fluorenylmethel-succinimidyl-carbonate method as described in example2. The Fmoc protected peptide is coupled to Doxorubicin using DIC asdescribed in example 14.

C. Synthesis of Thr-Thr-Asn-Leu-Doxorubicin

Deprotection of the Fmoc group is carried out by 20% piperidin in DMFfor 30 minutes with mixing at room temperature. TheThr-Thr-Asn-Leu-Doxorubicin is analyzed using reversed-phase HPLC andmass spectrometry as described in Example 4. The product gives solidpowder with molecular mass of 989 g/mol.

Example 16 Synthesis of Legumain Cleavable Prodrug 10 A. Synthesis ofLegumain Cleavable Peptide: Asn-Lys

The peptide cleavable by legumain was synthesized as described inexample 4. The first amino acid Fmoc-Lys(Mtt)-OH was coupled to Wangresin by using Dichlorobenzoylchloride method as described in example 4.

Boc-Asn(Trt)-OH was bound to the peptide-resin by subsequent steps ofdeprotection and coupling. The Lys protecting group Mtt was removed with1% TFA in DCM and monitored by O.D. at 460 nm.

B. Synthesis of Asn-Lys(Acetyl-5-Fluorouracil)

The drug 5-Fluorouracil-1-Acetic acid was coupled to peptidyl resin withDIC/HOBT in DMF. The resin was washed with DMF and DCM. Cleavage ofpeptide-drug was carried out by TFA with 10% H₂O. The TFA was evaporatedand the product was precipitated and washed withtert-Butyl-Methyl-Ether.

The Asn-Lys(Acetyl-5-Fluorouracil) was analyzed using reversed-phaseHPLC and mass spectrometry as described in Example 4.

Example 17 Tissue Culture Analysis of the Drug Combination

Human umbilical vein-derived endothelial cells (HUVEC) were preparedfrom umbilical veins and cultured as described previously. The HUVEcells were grown in M199 medium supplemented with 20% fetal calf serum,vitamins, 1 ng/ml hbFGF, and antibiotics.

Human melanoma cells (WW-94) were grown in 50% DMEM/50% F-12 mediumsupplemented with 10% fetal calf serum and antibiotics.

Human Breast cancer cells (MCF-7) were grown in DMEM medium supplementedwith 10% fetal calf serum and antibiotics.

To determine the biological activity of the drugs, HUVEC (normal cells)or melanoma (cancer) cells were seeded in 24-well dish at aconcentration of 10,000-20,000 cells/well, and increasing concentrationsof the prodrug 3-Peptide I-Chlorambucil (P-CAC) were added to the wells.As controls, either Cholorambucil or the peptide carrier was added underthe same experimental conditions. After 72 hours the cells were washedwith phosphate buffer saline (PBS), suspended with 0.5% EDTA/PBS, andcounted using cell coulter (Electronics ZM). Cell viability was verifiedby trypan-blue staining and hemocytometer analysis. IC50 indicates theconcentration of the drug measured at 50% of the cell death.

FIG. 4 shows that the peptide carrier is not toxic either to the HUVECor to melanoma cells. Chlorambucil is very toxic both to cancer andnormal cells (IC50 is 2 mM). The prodrug Peptide I-Chlorambucil isefficient as a chemotherapeutic agent in killing cancer cells with anIC50 of 2 mM (FIG. 4A). However, this prodrug is much less toxic to HUVEcells (IC50 of 60 mM)(FIG. 4B), i.e., 30 times less toxic to normalcells.

Example 18 MTT Assay for Estimation of the Toxicity of the Drugs

HUVEC or melanoma cells were seeded in 96-well ELISA dish at aconcentration of 10,000-20,000 cells/well, and various concentrations ofthe prodrug 1 were added to the wells as described in Example 17. As acontrol, Melphalan was added under the same experimental conditions.After 72 hours the medium was aspirated, and DMEM containing 5% FCS and0.5 mg/ml MTT (an indicator of cell viability) was added. The cells wereincubated for 2-4 hours at 37° C. and 5% CO2. At the end of theincubation the cells were washed with phosphate buffer saline (PBS), anddissolved with DMSO. The results were analysed using Techan ELISA readerequipped with a 570 nm filter.

FIG. 5 shows that Melphalan (♦) kills efficiently both cancer and normalcells. It also shows that the prodrug Peptide I-Melphalan (▪) is asefficient as the Melphalan itself in killing cancer cells (FIG. 5B), butit is less toxic to normal cells (FIG. 5A). The IC50 for Melphalan inHUVE cells and in melanoma cells is 0.1 mM, and 0.07 mM, respectively.The IC50 for the prodrug 1 in HUVE cells and in melanoma cells is 0.5 mMand 0.07 mM, respectively. These results show that this prodrug ofMelphalan is 7 times less toxic to normal cells.

Example 19 In-Vitro Release of the Methotrexate from PeptideI-Methotrexate

To find out whether proteases are secreted to the conditioned medium ofcancer cells, breast cancer cells or melanoma cells were grown tosub-confluence in a 10 cm dish. The cells were washed with PBS, andfurther incubated with serum free medium. HUVE cells were grown toconfluence at 10 cm dish. The cells were washed with M-199 medium, andconditioned with M-199 with 1% heat inactivated serum. The conditionedmedium was collected after 24 hours.

In-vitro release of Methotrexate from the peptide I-Methotrexate wascarried out in a liquid phase. The prodrug was dissolved in PBS, andincubated for up to 4 hours at 37° C. in the absence or presence of thecancer cells or HUVE cells (normal cells) conditioned medium. Therelease of the Methotrexate from the peptide I-Methotrexate wasmonitored by spectroscopic measuring of the free Methotrexate at 302 nm.

Example 20 In-Vitro Release of the Melphalan from Peptide III-Melphalan

In-vitro release of Melphalan from the peptide III-Melphalan was carriedout in a liquid phase as described in Example 19. The drug was dissolvedin PBS and incubated for 3 hours at 37° C. in the absence or presence ofcancer cells or normal cells conditioned medium. The release of theMelphalan from the peptide III-Melphalan was monitored by spectroscopicmeasuring of the free Melphalan at 254.

Example 21 In-Vitro Release of the Chlorambucil from the PeptideI-Chlorambucil

In-vitro release of Chlorambucil from the peptide carrier was carriedout in a liquid phase as described in Example 19. Peptide I-Chlorambucilwas dissolved in PBS, and incubated for 2 hours at 37° C. in the absenceor presence of purified MMPs, or in the presence of conditioned mediumof either human melanoma cells (HMCM) or human breast cancer cells(BCCM). To show that the GAGs can modulate the release of thechemotherapeutic agent from the peptide carrier the 2 hours incubationmentioned above was carried out in the absence or presence of 10 μg/mlheparin.

At the end of the incubation the reaction mixture was incubated withheparin-sepharose (30 minutes at 0° C.). The supernatant was collected,and the release of free chlorambucil was monitored by spectrophotometerat a wavelengh of 305 nm.

FIG. 6 shows that the chemotherapeutic agent can be released from thepeptide carrier either by purified MMPs, or by MMPs secreted by cancercells.

Addition of heparin to the reaction mixture inhibited the release of thechemotherapeutic agent from the peptide carrier, suggesting that GAGscan modulate the drug activity. For example, native proteoglycans suchas Serglycin can be used as a carrier for the drug, protecting it fromdegradation.

Example 22

In adult nude mice the following procedures are carried out undergeneral anesthesia (Ketamin 1 mg/10 g b.w.+0.01 mg/10 g b.w.):

A. A chronic I.V. access is opened by inserting a catheter to thejugular vein.B. S.C. Injection of 100 μl volume of human cancer cell suspension innormal saline containing 1×10⁶ cells. Tumor xenografts proliferatewithin 2-6 weeks in order to reach predetermined average size. The drugsare daily delivered by i.v. injections.The pharmacological agents in use are prodrugs of Melphalan with dosesof 40-150 mg/m² and prodrugs of Fluorouracil with doses of 300-450mg/m².

Example 23 Manufacture of a Medicament Containing Synthetic Peptides ofthe Invention

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the prodrugs described herein, or physiologicallyacceptable salts thereof, with other chemical components such asphysiologically suitable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of a compoundto an organism.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Pharmaceutical compositions may also include one or more additionalactive ingredients, such as, but not limited to, conventionalanti-migraine agents.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, grinding, pulverizing, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants for exampleDMSO, or polyethylene glycol are generally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. In addition enterocoating are useful as it is desirableto prevent exposure of the peptides of the invention to the gastricenvironment.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the peptides for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from a pressurized pack or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the peptide and a suitable powder base suchas lactose or starch.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active ingredients in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds, to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forreconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater, before use.

The compounds of the present invention may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount of acompound effective to prevent, alleviate or ameliorate symptoms of adisease of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Toxicity and therapeutic efficacy of the peptides described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ (the concentrationwhich provides 50% inhibition) and the LD₅₀ (lethal dose causing deathin 50% of the tested animals) for a subject compound. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,and all other relevant factors.

The following example is an illustration only of a method of treating asubject with a peptide according to the invention, in order to treat apathological condition associated with a solid tumor or a relatedcondition, and is not intended to be limiting.

The method includes the step of administering the prodrug, in apharmaceutically acceptable carrier as described above, to a subject tobe treated. The medicament is administered according to an effectivedosing methodology, preferably until a predefined endpoint is reached,such as a reduction or amelioration of the pathological condition in thesubject.

REFERENCES

-   Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J.,    Gallo, R. L. and Lose, E. J. (1992) Annu Rev. Cell Biol. 8, 365-393.-   Dano K, Behrendt N, Brünner N, Ellis V, Ploug M, Pyke C:    Fibrinolysis 1994, 8: 189-203.-   Fidler I. J. (1997) In: Devita V. T., et al. (Eds.) Cancer:    Principles and Practice of Oncology (5th edn). (pp. 135-152):    Lippincott-Raven.-   Gitay-Goren, H., Soker, S., Vlodaysky, I. and Neufeld, G. (1992) J.    Biol. Chem. 267, 6093-6098.-   Kuefner, U., Lohrman, U., Montejano, Y. D., Vitols, K. S., and    Hunnekens, F. M. (1989) Biochemistry 28: 2288-97.-   Massova I. et al. (1998) FASEB J., 12, 1075-1095.-   Menger et al. (1994) Bioconjugate Chem. 5, 162-166.-   Poltorak, Z., Cohen, T., Sivan, R., Kandelis, Y., Spira, G.,    Vlodaysky, I., Keshet, E., and Neufeld, G. (1997) J. Biol. Chem.    272, 7151-7158.-   Yamada, K. M. and Geiger, B. (1997) Curr. Opin. Cell Biol. 9, 76-85.-   Yayon, A., M. Klagsbrun, J. D. Esko, P. Leder, and D. M. Ornitz    (1991). Cell. 64: 841-848.

1. A prodrug comprising a conjugate of at least one antiproliferativedrug molecule covalently coupled directly or via a linker to a peptidemoiety, said peptide moiety being specifically cleavable by anasparaginyl protease abundant in or secreted by malignant cells, therebypreferentially releasing the antiproliferative drug molecule within orat the malignant cells by the cleaving action of the asparaginylprotease.
 2. The prodrug of claim 1, wherein the antiproliferative drugis selected from the group consisting of a cytotoxic, a cytostatic or achemotherapeutic drug.
 3. The prodrug of claim 1, wherein the conjugateis substantially pharmacologically inactive.
 4. The prodrug of claim 1,wherein the antiproliferative drug is selected from the group consistingof alkylating agents, antimetabolites, antitumor antibiotics, vincaalkaloids, epipodophyllotoxins, nitrosoureas, hormonal and antihormonalagents, and toxins.
 5. The prodrug of claim 1, wherein theantiproliferative drug is selected from the group consisting of:cyclophosphamide, chlorambucil, busulfan, Melphalan, Thiotepa,ifosphamide, Nitrogen mustard, methotrexate, 5-Fluorouracil, cytosinearabinoside, 6-thioguanine, 6-mercaptopurine, doxorubicin, daunorubicin,idorubicin, nimitoxantron, dactinomycin, bleomycin, mitomycin,plicamycin, epipodophyllotoxins vincristin, vinblastin, vindestin,Etoposide, Teniposide, carmustin, lomustin, semustin, streptozocin,adrenocorticorticoids, estrogens, antiestrogens, progestins, aromataseinhibitors, androgens, antiandrogens, dacarbazin, hexamethylmelamine,hydroxyurea, mitotane, procarbazide, cisplastin, and carboplatin.
 6. Theprodrug of claim 5, wherein the antiproliferative drug is selected froma group consisting of Melphalan, Methotrexate, Chlorambucil,Doxorubicin, and 5-fluorouracil.
 7. The prodrug of claim 1, wherein thepeptide cleavable by the asparaginyl protease consists of from two toabout fourteen amino acids at least one of which is asparagine.
 8. Theprodrug of claim 1, wherein the asparaginyl protease is legumain.
 9. Theprodrug of claim 8, wherein the peptide cleavable by legumain consistsof two to about fourteen amino acids comprising the amino acid sequenceX1-X2-X3, wherein X1 is selected from the group consisting of Pro, Thr,Asn, and Ala, X2 is selected from the group consisting of Thr, Pro, Asn,and Ala, and X3 is Asn.
 10. The prodrug of claim 9, wherein the peptidecleavable by legumain is selected from the group consisting of SEQ IDNO:9 to SEQ ID NO:32.
 11. The prodrug of claim 1 further comprising atleast one protecting group capable of preventing digestion of thepeptide moiety by nonspecific proteases.
 12. The prodrug of claim 1comprising a plurality of antiproliferative drug molecules.
 13. Theprodrug of claim 1, wherein the linker between the drug and the peptidecomprises any chemical compound which may be removed chemically,enzymatically or which decomposes spontaneously.
 14. The prodrug ofclaim 8, wherein the peptide cleavable by legumain consists of the aminoacid sequence selected from the group consisting of SEQ ID NO:33 to SEQID NO:38 inclusive.
 15. A pharmaceutical composition comprising as anactive ingredient a prodrug according to claim 1 together with apharmaceutically acceptable excipient or diluent.
 16. A method oftreating a subject suffering from a cancer malignancy comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition according to claim
 15. 17. The method ofclaim 16, wherein the pharmaceutical composition is administeredintravenously.
 18. A method for producing a prodrug, the methodcomprising the step of linking at least one antiproliferative drugmolecule and a peptide moiety to form the prodrug of claim 1, with thepeptide moiety being specifically cleavable by an asparaginyl proteaseabundant in or secreted by malignant cells.
 19. The method of claim 18,wherein the antiproliferative drug is selected from the group consistingof alkylating agents, antimetabolites, antitumor antibiotics, vincaalkaloids, epipodophyllotoxins, nitrosoureas, hormonal and antihormonalagents, and toxins.
 20. The method of claim 18, wherein the peptidemoiety cleavable by the asparaginyl protease consists from two to aboutfourteen amino acids at least one of which is asparagine.
 21. The methodof claim 18, wherein the asparaginyl protease is legumain.